Throughout this application various publications are referenced by full citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Pharmacological studies, and more recently gene cloning, have established that multiple receptor subtypes exist for most, if not all, neurotransmitters. The existence of multiple receptor subtypes provides one mechanism by which a single neurotransmitter can elicit distinct cellular responses. The variation in cellular response can be achieved by the association of individual receptor subtypes with different G proteins and different signalling systems. Further flexibility is provided by the ability of distinct receptors for the same ligand to activate or inhibit the same second messenger system.
Individual receptor subtypes reveal characteristic differences in their abilities to bind a number of ligands, but the structural basis for the distinct ligand-binding properties is not known. Physiologists and pharmacologists have attempted to specify particular biological functions or anatomical locations for some receptor subtypes, but this has met with limited success. Similarly, the biochemical mechanisms by which these receptors transduce signals across the cell surface have been difficult to ascertain without having well-defined cell populations which express exclusively one receptor subtype.
Dopamine receptors have been classified into two subtypes, D1 and D2, based on their differential affinities for dopamine agonists and antagonists, and their stimulation or inhibition of adenylate cyclase (for reviews, see Kebabian, J. W. and Calne, D. B. (1979), Nature 277, 93-96; Creese, I., Sibley, D. R., Hamblin, M. W., Leff, S. E. (1983), Ann. Rev. Neurosci. 6, 43-71; Niznik, H. B. and Jarvie, K. R. (1989), Dopamine receptors. in xe2x80x9cReceptor Pharmacology and Functionxe2x80x9d, eds. Williams, M., Glennon, R., and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). The D1 receptor of the central nervous system is defined as an adenylate cyclase stimulatory receptor. The location-of the prototypic D1 receptor is the bovine parathyroid gland, where dopamine agonists stimulate cAMP synthesis via adenylate cyclase, accompanied by parathyroid hormone release. Dopamine-stimulated adenylate cyclase activity and parathyroid hormone release are sensitive to both GTP and cholera toxin. This suggests that the D1 receptor is associated with a Gs guanine nucleotide binding protein. The D2 receptor, in contrast, inhibits adenylate cyclase activity, and appears to be the primary target of most neuroleptic drugs (Niznik, H. B. and Jarvie, K. R. (1989). Dopamine receptors, in xe2x80x9cReceptor Pharmacology and Functionxe2x80x9d, eds. Williams, M., Glennon, R., and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). The prototypic D2 receptor has been characterized in the anterior pituitary where it is associated with the inhibition of release of prolactin and alpha-melanocyte stimulating hormones. Recent work has shown that several different D1 and D2 receptor subtypes may be present in the mammalian nervous system (Andersen, P. H., Gingrich, J. A., Bates, M. D., Dearry, A., Falardeau, P., Senogles, S. E., and Caron, M. G. Trends in Pharmacolog. Sci. 11: 231 (1990)), which would suggest that a family of different proteins with pharmacological properties similar to the classically defined D1 and D2 receptors may exist.
Neuroleptics, in addition to their use as drugs to treat severe psychiatric illnesses, are high affinity ligands for dopamine receptors. Butyrophenones such as haloperidol and spiperone are antagonists specific for the D2 receptor, while the recently developed benzazepines such as SCH-23390 and SKF-38393 are selective for the D1 receptor (Niznik, H. B. and Jarvie, K. R. (1989), Dopamine receptors, in xe2x80x9cReceptor Pharmacology and Functionxe2x80x9d, eds. Williams, M., Glennon, R., and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). High affinity D1 and D2 selective ligands have conclusively distinguished these receptors and made feasible characterization of the receptors in the central nervous system and peripheral tissues with radioligand binding techniques. Two types of dopamine receptors, designated DA1 and DA2, have been identified in the cardiovascular system and are similar in their pharmacological characteristics to the brain D1 and D2 receptors (Niznik, H. B. and Jarvie, K. R. (1989), Dopamine receptors, in xe2x80x9cReceptor Pharmacology and Functionxe2x80x9d, eds. Williams, M., Glennon, R., and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). DA1 receptors have been described in renal, mesenteric, splenic, coronary, cerebral, and pulmonary arteries and vascular beds, where dopamine elicits relaxation of vascular smooth muscle. Activation of cardiovascular DA1 receptors appears to stimulate adenylate cyclase activity. DA2 receptors appear to be localized on preganglionic sympathetic nerve terminals that mediate inhibition of norepinephrine release. The molecular relationships among dopamine D1, D2, and DA2 receptors are unknown.
The need for improved selectivity in the leading D1 drug class, the benzazepines (e.g. SKF-38393, SCH-23390 and SCH-23982) recently became apparent when the strong cross-reactivity of these drugs with the serotonin 5-HT2 receptor family was uncovered. The 5-HT2 and 5-HT1C receptors display affinities ranging from 0.2 to 24 nM for SCH-23390 and SCH-23982 (Nicklaus, K. J., McGonigle, P., and Molinoff, P. B. (1988), J. Pharmacol. Exp. Ther. 247, 343-348; Hoyer, D. and Karpf, A. (1988), Eur. J. Pharmacol. 150, 181-184)), raising the possibility that behavioral and pharmacological effects ascribed to these drugs may, in fact, arise from serotonergic receptor interactions.
The dopamine D1 receptors belong to a family of receptors which are distinguished by their seven-transmembrane configuration and their functional linkage to G-proteins. This family includes rhodopsin and related opsins (Nathans, J. and Hogness, D. S., Cell 34:807 (1983)), the xcex1 and xcex2 adrenergic receptors (Dohlman, H. G., et al., Biochemistry 26:2657 (1987)), the muscarinic cholinergic receptors (Bonner, T. I., et al., Science 237:527 (1987)), the substance K neuropeptide receptor, (Masu, Y., et al., Nature 329:836 (1987)), the yeast mating factor receptors, (Burkholder, A. C. and Hartwell, L. H., Nucl. Acids Res. 13:8463(1985); Hagan, D. C., et al., Proc. Natl. Acad. Sci. USA 83:1418 (1986)); Nakayama, N. et al., EMBO J. 4:2643 (1985)), and the oncogene c-mas, (Young, et al., Cell 45:711 (1986)). Each of these receptors is thought to transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Dohlman, H. G., et al., Biochemistry 26:2657 (1987); Dohlman, H. G., et al., Biochemistry 27:1813 (1988); O""Dowd, B. F., et al., Ann. Rev. Neurosci., in press).
The D2 receptor was recently cloned by Civelli and colleagues (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C. A., Neve, K. A., and Civelli, O. (1989), Nature 336: 783-87). This event was soon followed by the discovery of an alternatively spliced form (termed D2A, D2long, D-2in, or D2(444)) that contains an additional 29 amino acids in the third extracellular loop of this receptor (Eidne, K. A. et al. (1989), Nature 342: 865; Giros, B. et al. (1989), Nature 342: 923-26; Grandy, D. K. et al. (1989), Proc. Natl. Acad. Sci. USA 86: 9762-66; Monsma, F. J. et al. (1989), Nature 342: 926-29; Chio, C. L. et al. (1990), Nature 343: 266-69; Stormann, T. M. et al. (1990), Mol. Pharmacol. 37: 1-6). A second dopamine receptor has been cloned which exhibits significant homology to the D2 receptor, both in amino acid sequence (75% transmembrane region identity) and in pharmacological properties (Sokoloff, P. et al. (1990), Nature 347: 146-51). This new receptor, termed D3, is encoded by an intron-containing gene. Unlike the D2 receptor, however, alternatively spliced isoforms of this receptor have yet to be observed. The D3 receptor has been shown to serve both as an autoreceptor and as a postsynaptic receptor, and has been localized to limbic areas of the brain (Sokoloff, P. et al. (1990), Nature 347: 146-51). Finally, an intronless gene, quite different in sequence and gene structure from the other two dopamine receptor genes, has been isolated and identified as a D1 dopamine receptor subtype (Sunahara, R. K. et al. (1990), Nature 347: 80-83; Zhou, Q.-Y. et al. (1990), Nature 347: 76-80; Dearry, A. et al. (1990), Nature 347: 72-76; Monsma, F. J. et al. (1990), Proc. Natl. Acad. Sci. USA 87: 6723-27). This D1 receptor is predominantly expressed in the rat striatum and olfactory tubercles, and has been shown to couple to stimulation of adenylate cyclase activity (Dearry et al. (1990) supra; Monsma et al. (1990) supra; Sunahara et al. (1990) supra; Zhou et al. (1990) supra. Available data on the G protein-coupled receptor superfamily suggests that the D1 receptor does not exhibit strong sequence homologies to the D2 receptor or the D3 receptor. In general, G protein-coupled receptors of the same neurotransmitter family exhibit closest structural homology to other family members that use the same second messenger pathway. For example, examination of the physiological second messenger pathways of the serotonergic, muscarinic and adrenergic receptors has led several researchers to the conclusion that these receptors can be classified into structurally homologous subtypes that parallel their second messenger pathways (Bylund, D. B. (1988), Trends Pharmacol. Sci. 9, 356-361; Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1988), Nature 334, 434-437; Liao, C.-F., Themmen, A. P. N., Joho, R., Barberis, C., Birnbaumer, M., and Birnbaumer, L. (1989), J. Biol. Chem. 264, 7328-7337; Hartig, P. R. (1989), Trends Pharmacol. Sci. 10, 64-69)). Interestingly, those receptors that couple to activation of adenylate cyclase appear quite distinct in structure from those that inhibit this enzyme activity.
Pharmacological and physiological data have emerged indicating the presence of further diversity within this receptor family. A D1 receptor that stimulates phosphoinositide (PI) hydrolysis in rat striatum has been described (Undie, A. S., and Friedman, E. (1990), J. Pharmacol. Exp. Ther. 253: 987-92) as well as an RNA fraction from the same tissue that causes dopamine-stimulated PI hydrolysis and intracellular calcium release when injected into Xenopus oocytes (Mahan, L. C. et al. (1990), Proc. Natl. Acad. Sci. USA 87: 2196-2200). In addition, two populations of peripheral D1 receptor have been described based on differential sensitivity to sulpiride and several other compounds (Andersen, P. H. et al. (1990), Eur. J. Pharmacol. 137: 291-93). Finally, pharmacological differences exist within different D1 receptor tissues that couple to adenylate cyclase-coupled D1 receptors. Biochemical and pharmacological data suggest further diversity in both the D1 and D2 receptor populations and indicate that additional dopamine receptor clones remain to be discovered (Andersen et al. (1990) supra).
This invention provides an isolated nucleic acid molecule encoding a human dopamine D1 receptor.
This invention also provides an isolated protein which is a human dopamine D1 receptor, an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIGS. 1A-1E (SEQ ID NO: 1)
This invention provides a vector comprising an isolated nucleic acid molecule encoding a human dopamine D1 receptor.
This invention provides a mammalian cell comprising a DNA molecule encoding a human dopamine D1 receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human dopamine D1 receptor can bind to a human dopamine D1 receptor which comprises contacting a mammalian cell comprising a DNA molecule encoding a human dopamine D1 receptor with the ligand under conditions permitting binding of ligands known to bind to the dopamine D1 receptor, detecting the presence of any of the ligand bound to the dopamine D1 receptor, and thereby determining whether the ligand binds to the dopamine D1 receptor.
This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human dopamine D1 receptor on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human dopamine D1 receptor on the surface of a cell with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the human dopamine D1 receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human dopamine D1 receptor.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human dopamine D1 receptor so as to prevent translation of the mRNA molecule.
This invention provides an antibody directed to the human dopamine D1 receptor.
This invention provides a transgenic nonhuman mammal expressing DNA encoding a human dopamine D1 receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human dopamine D1 receptor so mutated as to be incapable of normal receptor activity, and not expressing native dopamine D1 receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human dopamine D1 receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a dopamine D1 receptor and which hybridizes to mRNA encoding a dopamine D1 receptor thereby reducing its translation.
This invention provides a method of determining the physiological effects of expressing varying levels of human dopamine D1 receptors which comprises producing a transgenic nonhuman animal whose levels of human dopamine D1 receptor expression are varied by use of an inducible promoter which regulates human dopamine D1 receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of human dopamine D1 receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human dopamine D1 receptor.
This invention provides a method for diagnosing in a subject a predisposition to a disorder associated with the expression of a specific human dopamine D1 receptor allele which comprises a. isolating DNA from victims of the disorder, b. digesting the isolated DNA of step a with at least one restriction enzyme, c. electrophoretically separating the resulting DNA fragments on a sizing gel, d. contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human dopamine D1 receptor and labelled with a detectable marker, e. detecting labelled bands which have hybridized to the DNA encoding a human dopamine D1 receptor labelled with a detectable marker to create a band pattern specific to the DNA of victims of the disorder, f. preparing the subject""s DNA by steps a-e to produce detectable labeled bands on a gel, and g. comparing the band pattern specific to the DNA of victims of the disorder of step e and the subject""s DNA of step f to determine whether the patterns are the same or different and thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific human dopamine D1 receptor allele.
This invention provides a method of preparing the isolated dopamine D1 receptor which comprises inducing cells to express dopamine D1 receptor, recovering the receptor from the resulting cells, and purifying the receptor so recovered.
This invention provides a method of preparing the isolated dopamine D1 receptor which comprises inserting nucleic acid encoding dopamine D1 receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered.